ATF6α activation upon ER stress is prevented by sorafenib. A, the subcellular localization of FLAG-ATF6α was analyzed in HepG2 cells by immunofluorescence followed by wide-field fluorescence microscopy. Calnexin (CANX) is used as a marker for the ER. Scale bar corresponds to 25 μm. B, the number of cells containing nuclear-localized FLAG-ATF6α was quantified. Between 300 and 500 cells were analyzed for each condition, and results are represented as the mean ± SD. Student t test: *, P < 0.01. C, quantification of the mRNA expression of ATF6 responsive genes—Orp150, Herpud1, and BiP. Results shown are the mean ± SD of 3 experiments each with 2 replicates. Student t test: *, P < 0.05, compared with control. D, secretion of α1 antitrypsin (α1AT) in HepG2 cells was analyzed by immunoblotting (top). The amount of the corresponding intracellular α1AT was monitored in total cell lysate (bottom). Quantification of secreted α1AT was conducted on the immunoblots by densitometry. Results are shown as a percentage of control (CTL) and are the mean ± SD of 4 independent experiments. Student t test: *, P < 0.05; ***, P < 0.01 compared with control.

Sorafenib induces reversible Golgi complex fragmentation and autophagy in HepG2 cells. A, Golgi complex staining using anti-Giantin antibodies (green) and Hoechst 33342 as a nuclear marker (blue). Images were acquired by wide-field fluorescence microscopy. Scale bars correspond to 25 μm (right). The number of cells containing fragmented Golgi complex was quantified. Between 250 and 400 cells were analyzed for each condition, and results are represented as the mean ± SD (left). B, same as in A, but following sorafenib withdrawal. Student t test: *, P < 0.03; **, P < 0.01. C, HepG2 cells were transfected with a GFP-LC3 (green) construct for 24 hours and were then either left untreated (CTL) or treated with 10 μmol/L sorafenib for 8 hours. Cells were then analyzed by confocal microscopy. Scale bars represent 25 μm. D, total protein extracts of HepG2 cells transfected with a siRNA to ATG5 (for 48 hours) or treated with 40 mmol/L NH4Cl in the presence or not of 10 μmol/L sorafenib for 8 hours were subjected to immunoblotting against LC3. Tubulin is shown as a loading control. Results are representative of 3 experiments.

Tyrosine phosphorylation of p97/VCP is negatively regulated by sorafenib. A, HepG2 cells were either untreated (CTL) or treated with 10 μmol/L sorafenib, 5 μmol/L BpVphen, or 10 μmol/L sorafenib combined with 5 μmol/L BpVphen for 4 hours. p97/VCP was immunoprecipitated from total cell lysates and immunoblots against phospho-tyrosine (pY) and p97/VCP were conducted. The amount of pY in each sample is shown at the bottom of the immunoblots as a percentage of CTL. A representative experiment out of 4 is shown (left). Quantification of the 4 experiments by densitometry represented as the mean ± SD. Statistical significance is shown: *, P < 0.03; **, P < 0.01 (right). B, cells were treated as earlier and the presence of p97/VCP in the total membrane fraction was analyzed by immunoblotting. Calnexin is shown as a loading control. A representative experiment out of 3 is shown (left). Quantification of the 4 experiments by densitometry represented as the mean ± SD. Statistical significance is shown: *, P < 0.04; **, P < 0.01 (right). C, same as in A, but BpVphen was replaced by 20 μmol/L DBeQ. D, HuH7 cells were either not treated (1) or treated with 1 μmol/L (4) or 10 μmol/L (7) sorafenib for 2 hours, 5 μmol/L BpVphen (2), 20 μmol/L DBeQ (3) for 3 hours or the combination of 1 μmol/L sorafenib + 5 μmol/L BpVphen (5), 10 μmol/L sorafenib + 5 μmol/L BpVphen (8), and 1 μmol/L sorafenib + 20 μmol/L DBeQ (6). Cells were fixed and stained for Giantin (green) and Hoechst 33342 (blue). Images were acquired by confocal microscopy. Scale bars correspond to 10 μm.

Sorafenib-mediated cell toxicity occurs in part through p97/VCP. A, HepG2 (left) and HuH7 (right) cells were incubated for 48 hours with increasing concentrations of sorafenib or DBeQ or a combination of an increasing concentration of sorafenib and 20 μmol/L DBeQ, and the cellular viability was analyzed using sulforhodamine B staining. B, HepG2 (left) and HuH7 (right) cells were treated with 2 μmol/L sorafenib and 20 μmol/L DBeQ for 36 hours. Apoptosis was determined using Annexin V staining and is represented as mean ± SD representative of 3 independent experiments. Statistical significance is shown: *, P < 0.05; **, P < 0.01. C, HuH7 cells were treated with 2 μmol/L sorafenib and 20 μmol/L DBeQ or the combination of both for 16 hours. LC3 maturation was assessed using immunoblot with anti-LC3 antibodies. A blot representative of 3 independent experiments is shown. D, schematic representation of sorafenib-mediated regulation of cell death through p97/VCP and the secretory pathway. OD, optical density.

Additional Files

Supplementary Data

Supplementary Figure 1 -
PDF file - 877K, Figure S1. (A) Quantification of Xbp1 mRNA splicing shown in Figure 2A. (B) Wild-type HepG2 cells were
either not treated or treated with 10 μM Sorafenib for 2 h followed by staining for giantin, a Golgi complex marker, α-tubulin,
as a marker for the microtubules and Hoechst 33342 as a nuclear marker. (C) Cells treated as above were immunostained against
giantin and phalloidin was used to stain the actin cytoskeleton. Images were acquired by wide-field fluorescence microscopy.
Scale bars correspond to 25 μm.

Supplementary Figure 2 -
PDF file - 510K, Figure S2. (A) HepG2 cells were either not treated or treated with 10 μM Sorafenib for 4 h, after which
they were either fixed or Sorafenib removed from cells and fresh medium added for a further 2 h. Cells were then stained with
anti Giantin (green). Scale bars correspond to 25 μm. (B) The number of cells containing fragmented Golgi complex was quantified.
Between 200 and 400 cells were analyzed for each condition and results are represented as the means � SD. Student's t-test
*: p < 0.03, compared to control, #: p < 0.03 HepG2 mock transfected compared to IRE1-DN transfected cells.